Dopamine (DA) release in the cortex and basal ganglia is strongly implicated in modulation of CMS function[unreadable] and behavior and is thought to occur through a variety of potential secretory sites. Among these are axonal[unreadable] projections where small clear synaptic vesicles appear clustered in varicosities that resemble presynaptic[unreadable] terminals for typical fast-acting neurotransmitter secretion. Given that DA acts on much longer time scales[unreadable] than fast-acting neurotransmitters, the mechanism involved in controlling the presynaptic machinery may[unreadable] well be different than for those more typical "fast" synapses. Here we propose to examine details of the[unreadable] presynaptic vesicle cycle for these dopaminergic release sites. The long term objective of this proposal is to[unreadable] characterize the mechanism that control the presynaptic vesicle cycle for small clear dopaminergic[unreadable] veshicles. We will make use of technologies previously developed in the lab to examine many aspects of[unreadable] the molecular and biophysical nature of the presynaptic vesicle cycle in cortical and hippocampal cultures.[unreadable] These approaches rely heavily on optical techniques using exogenous organic probes such FM dye family[unreadable] members as well as genetically-encoded tags of presynaptic proteins that allow dynamic and quantitative[unreadable] information about the vesicle cycle to be obtained. These will be adapted to primary dissociated cell cultures[unreadable] of mid-brain neurons from the ventral tegmental area (VTA). We propose 3 specific aims to accomplish this[unreadable] initial characterization of the cell biological, physiological and biophysical aspects of the dopaminergic[unreadable] vesicle cycle. These include characterizing the properties of the vesicle pool in turns of depletion rates,[unreadable] replenishment rates, the sensitivity of pool turnover to stimulation at varied calcium concentrations, as well[unreadable] as the kinetics of endocytosis. Finally we will take advantage of the ability to detect dopamine sectretion[unreadable] directly using carbon-fiber amperometry to examine how details of the vesicle cycle impact neurotransmitter[unreadable] release.